The Investigation On The Mechanisms Of The Reciprocal Inhibition Between Obestatin And Insulin

With great changes in human lifestyle in recent years, the incidence of endocrine and metabolic diseases is rising yearly. Overweight, obesity and diabetes are very big threats to human health. Regulation of feeding behavior and metabolism is an important mechanism to maintain energy balance. When the intake is far more than the consumption, the energy balance is broken, which leads to fat accumulation and may result in overweight, obesity and complications of obesity such as diabetes. Investigation on the regulation mechanisms of feeding behavior and metabolism would benefit the prevention and cure of obesity and diabetes.Ghrelin was initially discovered by Kojima et al. in 1999 as an endogenous ligand for GHSR (growth hormone secretagogue receptor), but subsequent considerable and unequivocal evidence shows that it plays critical roles in the short-and long-term regulation of appetite and body weight. Ghrelin affects appetite and food intake as well as a diverse array of processes involved in energy expenditure and fuel utilization, all of which promote weight gain and fat accumulation. Exogenous ghrelin administration causes hyperphagia and obesity in rodents.Zhang and coworkers reported that ghrelin gene also encodes another 23-amino acid peptide, obestatin. The biological activity of obestatin depends on the amidation at its carboxyl terminus. Obestatin, though derived from the same peptide precursor, suppressed food intake, inhibited jejunal contraction, decreased body-weight gain, and antagonized the actions of ghrelin when both peptides are co-administered. These facts may suggest that the intricate balance of ghrelin and obestatin is important in the regulation of energy homeostasis and body weight control.Insulin can lower blood glucose concentration and promote the synthesis of glycogen, fat and protein. Insulin is closely related to fat and diabetes. Ghrelin can raise blood glucose level and inhibit insulin release. A recent prominent discovery in the obestatin field came from the effect of obestatin on insulin secretion. It is found that administration of exogenous obestatin suppresses insulin secretion in glucose-stimulated insulin secretion.To the best of our knowledge, there is little knowledge about the relationship between obestatin and insulin. Therefore, we attempted to investigate the relationship between obestatin and insulin as follows.1. The effect of glucose and insulin on plasma obestatin level was investigated using hyperglycemic clamp and hyperinsulinemic-euglycemic clamp.2. Diabetic rats were induced by a single intraperitoneal injection of 60 mg/Kg streptozotocin. Plasma obestatin and ghrelin levels and expressions of ghrelin/obestatin mRNA were investigated.3. INS-1 cells were cultured and used to investigate the effect of obestatin on normal and glucose stimulated insulin secretion. The mechanism was explored using blockers.4. The effects of obestatin on membrane potential, action potential, ATP-sensitive potassium currents and voltage-dependent calcium currents of INS-1 cells were investigated using perforated whole cell patch-clamp technique.Part 1 The effect of glucose and insulin on plasma obestatin levelObjectiveTo investigated the effect of glucose and insulin on plasma obestatin level.MethodsSD rats (not fasted, but 4 h after removal of food from cages) were assigned to 3 treatment groups:(A) saline-infused control. (B) hyperglycemic group infused with 20% dextrose for 3 h to goal blood glucose 16.7 mM. (C) hyperinsulinemic-euglycemic clamp (7.5 mU insulin/min per kg body weight, and variable rate 20% dextrose) to reach insulin concentrations similar to group B and glucose similar to group A. After baseline samples (150μl) were obtained, samples (5μl each) were obtained from the catheter in the jugular vein every 15 min. And samples (150μl each) were obtained every 60 min after treatment administration to measure obestatin and insulin using ELISA.ResultsIn control group, plasma obestatin level decreased slightly, but decreased promptly in hyperglycemic group and hyperinsulinemic-euglycemic group. At 120 minutes, plasma obestatin level were 0.860±0.180μg/L and 0.829±0.203μg/L respectively in hyperglycemic group and hyperinsulinemic-euglycemic group, compared with control group (1.188±0.242μg/L), the differences were significant (p<0.05, n=6).At 180 minutes, plasma obestatin level were 0.398±0.149μg/L and 0.369±0.132μg/L respectively in hyperglycemic group and hyperinsulinemic-euglycemic group, compared with control group (1.049±0.269μg/L), the differences were significant (p<0.01, n=6). The differences of plasma obestatin level between hyperglycemic group and hyperinsulinemic-euglycemic group were not statistically significant.ConclusionHigh concentration of plasma insulin reduced plasma obestatin levels, but high concentration of plasma glucose may has no effect on plasma obestatin levels.Part 2 Plasma obestatin and ghrelin levels and expression of ghrelin/obestatin mRNA in type 1 diabetic ratsObjectiveTo investigated plasma obestatin and ghrelin levels and expression of ghrelin/obestatin mRNA in type 1 diabetic rats.MethodsType 1 diabetic rats were induced by a single intraperitoneal injection of 60 mg/Kg streptozotocin (STZ). Plasma glucose levels and weights were measured weekly. Blood samples were collected to measure obestatin and insulin using ELISA, gastric tissues were collected to investigate the expression of ghrelin/obestatin mRNA by Real-time PCR.Results(1) Plasma obestatin levels remained unchanged at 2-6 weeks, but increased at 8 and 10 weeks, compared with control group, the differences were significant (p<0.05, n=5). (2) Plasma ghrelin levels remained unchanged at 2 weeks, and then increased at 4-10 weeks, compared with control group, the differences were significant (p<0.05, n=5). (3) The ghrelin/obestatin ratio first rose and then decreased. (4) The expression of ghrelin/obestatin mRNA in gastric tissues decreased at 10 weeks, compared with control group, the differences were significant (p<0.01, n=5). (5) The weight changes of diabetic rats were negatively correlated with plasma ghrelin levels (r=-0.833, p<0.05, n=5) and ghrelin/obestatin ratio (r=-0.824, p<0.05, n=5).ConclusionPlasma obestatin and ghrelin levels rose and expression of ghrelin/obestatin mRNA decreased in type 1 diabetic rats.Part 3 The effect of obestatin on glucose stimulated insulin secretion and its mechanismsObjectiveTo investigate the effect of obestatin on normal and glucose stimulated insulin secretion, and discuss the mechanisms.MethodsINS-1 cells was cultured and used to investigate the effect of obestatin on normal and glucose stimulated insulin secretion, and the mechanism was explored using blockers.Results(1) High concentration of glucose stimulated the secretion of insulin in INS-1 cells. (2) 10 nM and 100 nM obestatin suppressed the secretion of insulin stimulated by high concentration of glucose (p<0.01, n=6) and basal insulin secretion (p<0.05, n=6). (3) PTX and chelerythrine (PKC inhibitor) antagonized the effects of obestatin (p<0.01, n=6). Xestospongin C (IP3 receptor blocker), U73122 (PLC inhibitor) and neomycin (PLC/PLD inhibitor) had no significant effect.ConclusionOne mechanism by which obestatin decreased insulin secretion stimulated by glucose is by PTX-sensitive G proteins and PKC.Part 4 The effects of obestatin on membrane ion channels of INS-1 cellsObjectiveTo investigate the effects of obestatin on membrane ion channels of INS-1 cellsMethodsCultured INS-1 cells were used for perforated whole cell patch-clamp recording. We investigate the effects of obestatin on membrane potential, action potential, ATP-sensitive potassium currents (IKAP) and voltage-dependent calcium currents (ICaV).Results(1) Obestatin did not affect the membrane potential, but suppressed the amplitudes of action potential stimulated by glucose. Treatment with 10 and 100 nM obestatin for 5min, the amplitudes of action potential were 69.4±9.6%and 39.8±7.0% of the value before treatment, compared with the control group (93.1±3.6%), the differences were significant (p<0.01, n=5). (2) Obestatin had no significant effect on IKAP, but dose dependently inhibited Cav currents. After treatment with 10 and 100 nM obestatin for 5min, Cav currents were 60±9% and 45±6% of the value before treatment, compared with the control group (97±8%), the differences were significant (p<0.01, n=5).ConclusionObestatin suppressed Cav currents of INS-1 cells. Considering the results in part 3, we speculate one mechanism by which obestatin decreased insulin secretion stimulated by glucose is by activating PTX-sensitive G proteins, and then with help of membrane PKC, activated G protein suppressed the voltage-dependent calcium channels (VDCC).